Synthesis of new pyridothienopyrimidinone and pyridothienotriazolopyrimidine derivatives as pim-1 inhibitors

Abstract Three series of 2-arylpyridothieno[3,2-d]pyrimidin-4-ones 3a–j, pyridothienotriazolopyrimidines 6–8 and 4-imino-pyridothieno[3,2-d]pyrimidines 9a,b were prepared to improve the pim-1 inhibitory activity of the previously reported 2-arylpyridothieno[3,2-d]pyrimidin-4-ones. All the test compounds showed highly potent pim-1 inhibition with IC50 in the range of 0.06–1.76 µM. No significant difference was detected between the pim-1 inhibitory activity of the 4-pyrimidinone and the 4-imino (=NH) or the cyclised triazolopyrimidine derivatives. The most active compounds were tested for their cytotoxic activity on MCF7 and HCT116 and showed potent activity on both the cell lines.


Introduction
The proviral integration site for Moloney murine leukaemia virus-1 (known as pim-1) is a serine/threonine kinase that controls many cellular functions including cell cycle, cell differentiation, cell survival, apoptosis and drug resistance [1][2][3] . High levels of pim-1 kinase are associated with many types of cancer such as myeloid leukaemia, breast cancer and prostatic cancer [1][2][3][4] . The identification of the role of pim-1 in controlling the growth of cancer stem cells and promotion of multiple drug resistance added more to the importance of developing potent pim-1 inhibitors as anticancer agents that can overcome the drug resistance developed by cancer stem cells 1,5 .
Recently, we had reported the identification of pyridothienopyrimidin-4-one derivatives IV as potent pim-1 inhibitors 9 . These derivatives were developed through structure rigidification strategy via ring closure of their precursors thieno [2,3-b]pyridines III 10 to ensure the presence of the carbonyl group at proper orientation for binding with the enzyme. Indeed, our efforts led to significant improvement in the enzyme inhibitory activity as well as the cytotoxic activity. The most potent inhibitors were the 2-aryl-2,3dihydro derivatives Va-c that exhibited pim-1 inhibition in the range of 1.18-1.97 mM 9 . However, the aryl groups used in that study were all bearing ortho substitution to mimic the structure of the previously published benzofuropyrimidinone derivative VI 11 ( Figure 2).
In continuation to these efforts, we reported herein the SAR study of the effect of substitution on different positions of the aryl group on the pim-1 inhibitory activity. Thus, para substitution, disubstitution and trisubstitution on the phenyl ring were all investigated (compounds 3a-j). Besides, the effects of isosteric replacement of the C¼O with ¼ NH to give 4-imino derivatives (9a,b) or cyclisation into pyridothienotriazolopyrimidines (6-8) on pim-1 inhibition were investigated. It is noteworthy that this is the first published work describing the pim-1 inhibitory activity of pyridothienotriazolopyrimidine derivatives ( Figure 3).
All the new compounds were tested for their pim-1 enzyme inhibitory activity and the most active compounds were further tested for their anti-proliferative activity using two different cell lines MCF7 and HCT116.

General notes
Stuart SMP20 apparatus was used to determine the melting points and they were uncorrected. The IR spectra were recorded on Shimadzu IR 435 spectrophotometer (Kyoto, Japan) and the values were represented in cm À1 . The 1 H NMR and 13 C NMR spectra were recorded on Bruker 400 and 100 MHz spectrophotometer, respectively. TMS was used as an internal standard and the chemical shifts were recorded in ppm on d scale. Both IR and NMR spectra were carried out at Faculty of Pharmacy, Cairo University, Cairo, Egypt. The electron impact mass spectra were recorded on Thermo Scientific ISQLT single quadrapole mass spectrometer. Both mass spectra and elemental analyses were carried out at the regional centre for mycology and biotechnology, Al-Azhar University, Cairo, Egypt. All reagents and solvents were purified and dried by standard techniques. 3-Amino-5-bromo-4,6-dimethylthieno [2,3-b]pyridine-2carbonitrile (1) and 3-amino-5-bromo-4,6-dimethylthieno[2,3b]pyridine-2-carboxamide (2) were prepared according to the published methods 9,12 .

b)
A mixture of compound 5 (0.64 g, 0.002 mol) and acetic anhydride or 2,2,2-trifluoroacetic anhydride (4 ml) was heated under reflux for 5 h and then allowed to cool. The reaction mixture was poured onto ice-cold water (100 ml) and the product was filtered, dried and crystallised from acetic acid.

Pim-1 kinase inhibitory activity
The kinase inhibitory activity of the synthesised compounds was determined using Human proto-oncogene serine/threonine-protein kinase pim-1 (PIM1) ELISA Kit (catalog #MBS7573). All the compounds were tested for their inhibitory activity against pim-1 kinase at 0.2, 1, 5 and 25 mM using Staurosporine as a reference standard. The results were displayed in terms of percent inhibition and IC 50 . Table 1 and Figure 4 show the obtained results.

In vitro cytotoxic activity
Cell culture protocol Cell line cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cell lines used in this study were human breast adenocarcinoma (MCF7) and human colon adenocarcinoma (HCT116). The cells were cultured using DMEM (Invitrogen/Life Technologies) supplemented with 10% FBS (Hyclone), 10 mg/mL insulin (Sigma) and 1% penicillin-streptomycin. All of the other chemicals and reagents were from Sigma or Invitrogen.
Plate cells (cells density 1.2-1.8 Â 10,000 cells/well) in a volume of 100 mL complete growth medium and 100 mL of the test compound per well were prepared in a 96-well plate for 24 h before the MTT assay. The culture medium was removed to a centrifuge tube. The cell layer was rinsed with 0.25% (w/v) Trypsin 0.53 mM EDTA solution to remove all traces of serum which contains Trypsin inhibitor. Then, 2-3 ml of Trypsin EDTA solution was added and the cells were observed under an inverted microscope until the cell layer is dispersed (usually within 5-15 min.). A volume of 6 to 8 ml of complete growth medium was added and the cells  Table 2 and graphically represented in Figure 5.

Results and discussion
Chemistry Schemes 1 and 2 outline the synthesis of the target compounds.
In the current work, 3-amino-5-bromo-4,6-dimethylthieno [ Compound number IC50 Figure 4. IC 50 of the synthesised compounds in mM on pim-1 kinase.    Finally, the reaction of compound 5 with different aldehydes in glacial acetic acid furnished N-(substituted benzylidene)-4-iminopyridothienopyrimidin-3-amines 9a,b. Their IR spectra indicated the disappearance of NH 2 band, while their 1 H NMR spectra revealed the presence of singlet signal of N¼CH proton as well as the introduced aromatic protons.

Pim-1 kinase inhibitory activity
All the compounds were tested for their ability to inhibit pim-1 kinase at 0.2, 1, 5 and 25 mM using Staurosporine as a reference compound and the results in terms of percentage inhibition and IC 50 were displayed in Table 1 and represented graphically in Figure 4.
Upon designing the target pyridothienopyrimidinones, it was assumed that the substituent at position 2 of the ring occupied a solvent exposed area in pim-1 kinase active site and thus can accommodate various groups. Introduction of H-bonding and hydrophilic groups (like OH and methoxy groups) on the aryl ring especially at meta and para positions seemed to make extra Hbonding to the enzyme active site which might account for the higher kinase inhibitory activity exerted by compounds 3e and 3g.
On the other hand, SAR study of the pyridothienotriazolopyrimidines 6-8 indicated the following remarks: The unsubstituted triazole derivative 6 showed potent pim-1 inhibition with IC 50 ¼ 1.08 mM. Substitution with methyl group in compound 7a reduced the inhibitory activity. However, substitution with CF 3 group in compound 7b or C 2 H 5 OCOCH 2 moiety in compound 8 improved the inhibitory activity significantly.
Finally, the 4-imino derivative 9a, b showed also potent pim-1 inhibitory activity with IC 50 ¼ 0.52 and 0.22 mM, respectively. It is noteworthy that no significant difference was detected between the pim-1 inhibitory activity of the 4-pyrimidinone and the 4-imino (¼NH) or the cyclised triazolopyrimidine derivatives.

In vitro cytotoxic activity
The most active pim-1 inhibitors in this work (compounds 3b, 3c, 3e, 3g, 3j, 7b and 8) were tested for their cytotoxic activity against MCF7 and HCT116 cell lines using MTT method 13,14 . The results in terms of IC 50 in mM are given in Table 2 and represented graphically in Figure 5.
All the compounds showed potent cytotoxic activity on both cell lines. The cytotoxic activity on MCF7 cell line ranged between 0.22 and 1.84 mM. Five compounds (3c, 3e, 3g, 3j and 8) exerted potent cytotoxic activity with IC 50 values below 0.5 mM. Whilst, the cytotoxic activity on HCT116 ranged between 0.37 and 4.82 mM.
Compound 3e [the 2-(3,4-dihydroxyphenyl) derivative] displayed the most potent cytotoxic effect on both cell lines with IC 50 values of 0.22 and 0.37 mM, respectively. These results were consistent with its high kinase inhibitory activity (IC 50 ¼ 0.06 mM). While compound 3b showed the least potent cytotoxic activity on both cell lines with IC 50 values of 1.84 and 2.74 mM, respectively.
Notably, compounds 3b and 3e exhibited the same pim-1 inhibitory activity with IC 50 of 0.06 mM. The dramatic variation in their cytotoxic activity might be explained by examining their calculated log p values 15 ( Table 2). The calculated log P of compound 3b was 5.02, while that of 3e was 3.24.
It is noteworthy that significant improvement was observed in the cytotoxic activity of the tested compounds compared to their precursors Va-c. The latter cytotoxicity range was between 30 and 180 mM on the same cell lines 9 .

Conclusions
In this work, series of 2-arylpyridothieno[3,2-d]pyrimidin-4-ones 3a-j, pyridothienotriazolopyrimidine 6-8 and 4-imino-pyridothieno [3,2-d]pyrimidines 9a,b were prepared to improve the pim-1 inhibitory activity of the previously reported 2-arylpyridothieno [3,2-d]pyrimidin-4-ones. The lead optimisation strategies used were: isosteric replacement of C¼O by ¼NH and ring closure of the imino derivatives into triazolo ring. Besides, a brief SAR study of the substitution on the 2-aryl moiety was done. All the test compounds showed highly potent pim-1 inhibition with IC 50 in the range of 0.06-1.76 mM. Compounds 3g (3-OCH 3 -4-OH derivative) and 3e (3,4-dihydroxy derivative) were the most potent pim-1 inhibitors in this study with IC 50 of 0.08 and 0.06 mM, respectively. No significant difference was detected between the pim-1 inhibitory activity of the 4-pyrimidinone and the 4-imino (¼NH) or the cyclised triazolopyrimidine derivatives. The most active compounds were tested for their cytotoxic activity on two cell lines [MCF7 and HCT116] using MTT method. All the compounds showed potent cytotoxic activity on both cell lines but compound 3e [the 2-(3,4dihydroxy) derivative] displayed the most potent cytotoxic effect on both cell lines with IC 50 values of 0.22 and 0.37 mM and these results were consistent with its high kinase inhibitory activity (IC 50 ¼ 0.06 mM). A remarkable improvement in the cytotoxic activity was also noticed compared to the previously reported pyridothienopyrimidinones. Further work on pyridothienopyrimidine scaffold is still needed to obtain more potent pim-1 inhibitors and to improve the physicochemical properties of these derivatives.